Lipopolysaccharides (LPS; also referred to as endotoxins) are present in the cell walls of Gram-negative bacteria. When LPS is presented to a vertebrate body it stimulates the innate and cellular immune responses in a wide variety of cell types. The production of cytokines and chemokines (such as TNF's, various interleukines, interferons and others) will attract and activate cells of the immune system, which may culminate ultimately in an LPS induced systemic inflammatory response syndrome (SIRS) under certain conditions.
LPS or endotoxins are toxic to most mammals and regardless of the bacterial source, all endotoxins produce the same range of biological effects in the animal host. The injection of living or killed Gram-negative cells, or purified LPS, into experimental animals causes a wide spectrum of non-specific pathophysiological reactions such as: fever, tachycardia, tachypneu, hyper or hypothermia, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, organ dysfunction and may even result in shock and death.
Injection of small doses of endotoxin results in a proinflammatory response in most mammals, but the dose response range and steepness thereof varies significantly with the species and even within species may differ significantly depending on e.g. LPS-tolerance. The sequence of pro-inflammatory events follows a regular pattern (inflammatory cascade): (1) latent period; (2) physiological distress (diarrhea, prostration, shock); and in case of severe septic shock and multiple organ failure may result in (3) death. How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal.
The physiological effects of endotoxin are mainly mediated by the lipid A-moiety of LPS. Since Lipid A is embedded in the outer membrane of bacterial cells, it only exerts its toxic effects when released from multiplying cells in a soluble form, or when the bacteria are lysed as a result of autolysis, complement and the membrane attack complex (MAC), ingestion and killing by phagocytes, or killing with certain types of antibiotics. LPS released into the bloodstream can be neutralised by many blood components to a certain degree, amongst which several plasma lipids and proteins, among which LPS-binding proteins. The LPS-binding protein complex interacts with CD14 and Toll like receptors on monocytes and macrophages and through other receptors on endothelial cells. In monocytes and macrophages three types of events are triggered during their interaction with LPS:
Firstly, production of cytokines, including IL-1, IL-6, IL-8, tumor necrosis factor (TNF) and platelet-activating factor. These in turn stimulate production of prostaglandins and leukotrienes. These are powerful mediators of inflammation and septic shock that accompanies endotoxin toxemia. LPS activates macrophages to enhanced phagocytosis and cytotoxicity. Macrophages are stimulated to produce and release lysosomal enzymes, IL-1 (“endogenous pyrogen”), and tumor necrosis factor (TNFalpha), as well as other cytokines and mediators.
Secondly, activation of the complement cascade. C3a and C5a cause histamine release (leading to vasodilation) and effect neutrophil chemotaxis and accumulation. The result is inflammation.
Finally, activation of the coagulation cascade. Initial activation of Hageman factor (blood-clotting Factor XII) can activate several humoral systems resulting in coagulation: a blood clotting cascade that leads to coagulation, thrombosis, acute disseminated intravascular coagulation, which depletes platelets and various clotting factors resulting in internal bleeding and also activation of the complement alternative pathway (as above, which leads to inflammation). Plasmin is activated which leads to fibrinolysis and hemorrhaging and kinin activation releases bradykinins and other vasoactive peptides which causes hypotension. The net effect is induction of inflammation, intravascular coagulation, hemorrhage and shock.
LPS also acts as a B cell mitogen stimulating the polyclonal differentiation and multiplication of B-cells and the secretion of immunoglobulins, especially IgG and IgM.
The physiological activities of LPS are mediated mainly by the Lipid A component of LPS. Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system. During infectious disease caused by Gram-negative bacteria, endotoxins released from, or part of, multiplying cells have similar effects on animals and significantly contribute to the symptoms and pathology of the disease encountered. The primary structure of Lipid A has been elucidated and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO4 groups, the acyl chains, and also the KDO-containing inner core of the LPS molecule.
Alkaline phosphatase (AP), has been described earlier as a key enzyme in the dephosphorylation of LPS (endotoxin) under physiological conditions both in vitro and in vivo as a natural response to detoxify and neutralise LPS (U.S. Pat. No. 6,290,952, Poelstra et al., Am J Pathol. 1997 October; 151(4):1163-9).
Reports on the enzyme activity of AP involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. Endotoxins are molecules that contain several phosphate groups and are usually present in the extracellular space. AP is able to dephosphorylate this bacterial product at physiological pH levels, by removing phosphate groups from amongst others the toxic lipid A moiety of LPS. As phosphate residues in the lipid A moiety determine the toxicity of the molecule, the effect of the AP inhibitor levamisole in vivo using a septicemia model in the rat confirmed the specificity of AP for LPS containing phosphate groups (Poelstra et al., 1997). The results demonstrated that inhibition of endogenous AP by levamisole significantly reduces survival of rats intraperitoneally injected with E. coli bacteria, whereas this drug does not influence survival of rats receiving a sublethal dose of the gram-positive bacteria Staphylococcus aureus, illustrating a crucial role for this enzyme in host defense. The effects of levamisole during gram-negative bacterial infections and the localization of AP as an ecto-enzyme in most organs as well as the induction of enzyme activity during inflammatory reactions and cholestasis is in accordance with such a protective role.
The prime source of LPS exposure in the human body are the gram negative microorganisms that live within the human digestive or gastrointestinal (GI) tract. There are far more bacteria in the digestive system than there are on the skin or other parts of the body, making the GI tract and GI mucosa the main route of entry for LPS into the circulation. An average adult carries about 100 trillion bacteria in the intestines, most of which locate in the colon, contributing to 1-1.5 kg of his body weight. There are more than 400 species of bacteria found in the digestive system. These include both beneficial (commensal) and potentially harmful (pathogenic) species, which continually compete to maintain a well-balanced intestinal flora.
Mucosal surfaces, and in particular (but not limited to) the intestinal mucosa, are exposed to this wide variety of commensal and potentially pathogenic bacteria, among which many gram negative endotoxin/LPS producing, Gram-negative bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, Helicobacter, Chlamydia and other leading pathogens. The intestinal epithelium is of particular importance as it forms a dynamic barrier that regulates absorption of nutrients and water and at the same time restricts uptake of microbes and other noxious materials such as LPS from the gut lumen.
It is well established that a major fraction of LPS influx from the lumen of the gut through the mucosal lining into the circulation of a vertebrate body is mediated through chylomicrons (Harris et al., 1998, 2000, 2002). Coincidental with ingestion of lipids and chylomicron introduction in circulation, capable of carrying LPS, a significant increase in lymphatic AP derived from the GI-tract is reported (Nauli et al., 2002). LPS-influx through the GI-barrier is increased normally with a saturated fat-rich diet. LPS inserts with its lipid A acyl chain into lipoprotein phospholipids. Thereby LPS passes the intestinal barrier by co-migrating with chylomicrons, that are taken up predominantly at the small intestines ileum (Harris et al., 2002). After a fat rich food intake a significant rise of glycosyl-phosphatidyl-inositol (GPI)-anchored AP complexed to lipoproteins is detected in lymph as well (Nauli et al., 2003).
The physiological roles of—and the interpretation of AP serum levels are not clear, but a role in detoxification of LPS has emerged from current research. The co-presence of both AP and LPS in chylomicron rich fractions suggest a role for AP in dephosphorylating the gut derived-LPS already at close vicinity. Detoxification can take place both in the intestinal lumen or en-route to or upon presentation to the liver, specifically in this context to Kuppfer cells and the hepatocytes, which clear the chylomicrons from circulation.
Increased serum AP levels are associated with hepatic damage. Upon an endotoxin insult, circulatory AP is redirected to hepatocytes, thereby reducing circulating AP levels initially (Bentala et al., 2002) through receptor-mediated uptake (asialo-glycoprotein receptor). Hepatocytes also remove the LPS-loaded chylomicrons (Harris et al., 2002) rapidly from circulation with a half life of 5-10 minutes. LPS is next removed through biliary excretion, thereby preventing Kupffer cells, being a major target for circulating LPS to become activated (Harris, 2002). Bentala et al., 2002, showed that Kupffer cells accumulate AP in LPS-insulted animal models as well. This may imply that under normal conditions Kuppfer cells will not be activated since LPS (lipidA moiety), or its derivative MPLS (MPLA, derivative from Lipid A), is primarily presented to hepatocytes through a lipoprotein receptor and next is removed via biliary secretion. However under conditions with excess LPS, Kupffer cells are activated through a TLR-4 (LPS) receptor.
A wide array of animals have AP and several other entities present to counteract a (bacterial) insult, either local or systemic, induced or available as guard/watchdog function. Amongst others activated neutrophils or macrophages express a wide array of inflammatory mediators destined to neutralise the insult. Moieties like, but not restricted to LPS binding protein (LBP), CD14, Apo-E, VLDL, HDL, albumin, immunoglobulin and AP all have been described to serve this function. When such an insult however is not overcome, e.g. in case of a severe Gram negative or positive insult, resulting inflammatory mediators may initiate a systemic inflammatory response syndrome (SIRS).
It was postulated that AP is consumed as a consequence of its catalytic action towards LPS (Poelstra et al., 1997). This implies that subsequently normal levels are to be restored through a controlled mechanism. In patients suffering from septicaemia, it has been observed that increased serum AP may be preceded by reduced AP serum levels (Manintveld and Poelstra, patent application EP 989626940) and that circulating AP would be cleared from circulation upon LPS interaction (Bentala et al., 2002). The increase in subsequent AP-levels therefore may be a feedback mechanism in response to this AP reduction. A mechanism for such a LPS/AP responsiveness has not been depicted to-date.
In inflammatory processes (temporary) increases are found for serum AP. In the context of this invention such an increase of AP is regarded as a natural response of the innate immune system to an LPS insult to tackle these insults and restore natural balance. Increased AP plasma levels are the result of massive shedding of AP from hepatocytes in response to the LPS insult. It has been observed that LPS induces Phospholipase-D activity (Locati et al., 2001) which in turn has been reported to act upon GPI anchored proteins, amongst which AP (Deng et al., 1996) and e.g. CD14, thereby effectively shedding the proteins into circulation (Zhang F et al., 2001, Locati M. et al., 2001).
Circulating plasma AP—predominantly anchorless livertype AP (Ahn et al., 2001)—may thus already have exerted its LPS detoxificating activity at the plasma membrane surface and is subsequently freed from the hepatocyte membrane into circulation prior to its subsequent elimination from circulation by e.g. the asialo glycoprotein-route.
AP exerts its catalytic activity towards LPS primarily in the vincinity of a membrane, possibly in so-called lipid rafts (drm or detergent-resistant membrane fraction) where it has been reported to reside. Several publications favor such a catalytic activity of AP at a membrane surface, either presented at the tissue level or released into circulation like with circulating liver plasma membrane fragments (LPMF) (e.g. Deng et al., 1996). The increased AP levels observed in chronically inflamed patients may be caused by the suboptimal detoxification of the gut-derived influx of LPS, which is often enhanced under pathological conditions prior to mobilization of hepatic AP.
The treatment of inflammatory diseases accounts for a substantial percentage of the gross medical cost in developed countries and the incidence of these inflammatory diseases is continuously rising due to key factors like ageing of the population and an increasing number of patients having suppressed immune systems as a consequence of medication and treatment of a wide array of diseases like heart disease, auto-immunity disorders and allergies, organ transplantations, cancer chemo- or radiotherapy and infectious diseases like AIDS. To a certain extent these diseases relate to an influx of bacterial LPS. The influx of LPS is often enhanced by a medical condition of a subject, causing an inflammatory process by a malfunctioning or non-balanced innate immune system, which constitutes the first line of defense against e.g. microbial insults, in particular from LPS/endotoxin producing bacteria.
The current invention is aimed at providing new methods and compositions for the detoxification, neutralisation or complexation of LPS in situ at mucosal tissues in body cavities before LPS can pass through the mucosal layer and enter the circulation where it would elicit toxic effects and/or an inflammatory response.